Thermo Fisher Scientific Dynabeads Protein A Immunoprecipitation Kit User guide

Type
User guide
Kit contents
Component Volume
Dynabeads Protein A 2 mL
Ab Binding & Washing Buffer 16 mL
Washing Buffer 28 mL
Elution Buffer 1 mL
Dynabeads Protein A kit contains
sufcient reagents for 40 reactions. The
magnetic beads are at a concentration of
30mg/mL in phosphate buffered saline
(PBS), pH 7.4, with 0.01% Tween-20
and 0.09% sodium azide as a
preservative.
Caution: Sodium azide may react with
lead and copper plumbing to form
highly explosive metal azides.
Note: Read the Safety Data Sheets
(SDSs) for additional information on
buffers.
Product description
The kit contains Dynabeads Protein
A beads, as well as buffers for
binding, washing, and elution steps
for immunoprecipitation of proteins,
protein complexes, protein-nucleic acid
complexes, and other antigens.
Dynabeads Protein A
Immunoprecipitation Kit
For research use only. Not for use in diagnostic procedures.
Catalog No. 10006D Store at 2˚C to 8˚C
Publication No. MAN0017347 Rev. A.0
Antibodies (Ab) are added to the
Dynabeads Protein A, and bind to the
magnetic beads via their Fc-region during
a short incubation. The tube is placed on a
magnet, and the beads adhere to the side
of the tube facing the magnet, allowing
easy removal of the supernatant.
The bead-bound Ab is then used for
immunoprecipitation of the target antigen
(Ag). Bound material is easily collected
utilizing the unique magnetic properties of
the Dynabeadsmagnetic beads.
The Dynabeads Protein G kit can also be
used as part of an automated workow
on the KingFisher platform. Protocols for
the KingFisher Duo Prime System (up to
24 samples/run) and KingFisher Flex
System (up to 96 samples/run), go to
thermosher.com/kingsher.
Required materials
DynaMagMagnet (See
thermosher.com/magnets for
recommendations).
Sample mixer allowing tilting and
rotation of tubes (e.g., HulaMixer
Sample Mixer).
General guidelines
Dynabeads Protein A has a binding
capacity of ~8 µg of human IgG
per mg of beads. The amount
of Ab captured depends on the
concentration of beads and Ab in the
starting sample, as well as the type of
immunoglobulin being bound.
The choice of primary antibody is the
most important factor for successful
target Ag capture. Some antibodies
exhibit reduced Ag-binding efciency
for immunoprecipitation, even though
good results are seen with other
immunological assays. The afnity of
different antibody types to Protein A is
shown in Table 1.
For low-afnity antibodies or samples with low antigen concentration, pre-
incubate the sample and antibody (indirect technique) before bead capture
to improve binding kinetics for the antibody and minimize non-specic
binding. This approach is also recommended when working with protein/
nucleic acid complexes, e.g., ChIP.
Due to fast binding kinetics, an antibody only needs to be incubated with the
Dynabeadsmagnetic beads for 10 minutes for sufcient antibody capture.
Increase incubation time of the magnetic bead-Ab complex with the target
antigen to 20–120 minutes to increase yield for low afnity antibodies or
samples with low antigen concentration, although this may lead to increased
non-specic binding.
For sensitive proteins and phosphorylation studies, perform the isolation
protocol and elution at 2–8°C, to avoid protein complex dissociation and
minimize enzymatic activity.
Protocol
The protocol is a general guideline for immunoprecipitation. Optimization may
be required for each antibody (Ab) and target antigen (Ag). The protocol uses
50 µL of Dynabeads Protein A, but this may be scaled up or down as required.
Prepare Dynabeads magnetic beads
1. Resuspend Dynabeads magnetic beads in the vial (vortex >30 sec or tilt and
rotate 5 minutes).
2. Transfer 50 µL (1.5 mg) Dynabeads magnetic beads to a tube.
3. Place the tube on the magnet to separate the beads from the solution, and
remove the supernatant.
4. Remove the tube from the magnet.
5. Proceed directly to "Bind antibody".
Bind antibody
1. Add your antibody (typically 1–10 μg) diluted in 200 µL of Ab Binding and
Washing Buffer to the magnetic beads from step 4 in “Prepare Dynabeads
magnetic beads”. The optimal amount of Ab depends upon the individual
Ab used.
2. Incubate with rotation for 10 minutes at room temperature.
3. Place the tube on the magnet and remove the supernatant.
4. Remove the tube from the magnet and resuspend the magnetic bead-
Ab complex in 200µL Ab Binding and Washing Buffer. Wash by gentle
pipetting.
Note: Ab-conjugated magnetic beads can be stored in Ab Binding and
Washing Buffer to prevent aggregation.
5. Proceed to “Immunoprecipitate target antigen”.
(Optional) Crosslink antibody
To avoid co-elution of your antibody, crosslink your antibody to the Dynabeads
magnetic beads before immunoprecipitation. Use the crosslinking reagent
DSS (disuccinimidyl suberate). For further information and procedure, visit
thermosher.com/proteincrosslinking.
Immunoprecipitate target antigen
1. Place the tube (from step 4 of “Bind antibody”) on the magnet and remove
the supernatant.
2. Add your sample containing the antigen (typically 100–1,000 µL) and gently
pipette to resuspend the magnetic bead-Ab complex.
3. Incubate with rotation for 10 minutes at room temperature to allow the
antigen to bind to the magnetic bead-Ab complex.
Note: Depending on the afnity of the antibody, it may be necessary to
increase incubation times for optimal binding.
Figure 1: Principle of immunoprecipitation of
antigen using Dynabeads Protein A.
Dynabeads ProteinA
Bind antibody
(Crosslink antibody optional)
Y
Immunoprecipitate
target antigen
Y
Elute target antigen
Y
++
For support visit thermofisher.com/support or
thermofisher.com
14 September 2017
Corporate entity: Life Technologies | Carlsbad, CA 92008 USA | Toll Free in USA 1.800.955.6288
©2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its
subsidiaries unless otherwise specified.Tween is a registered trademark of Croda Americas, PLC.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.
Ig origin Affinity for Protein A
Human IgG1,2,4 +++
Human IgD
Human IgA, E, M +
Human IgG3 +
Mouse IgG1 +
Mouse IgG2, 2b, 3 +++
Mouse IgM +
Rat IgG1 +
Rat IgG2a
Rat IgG2b
Rat IgG2c +++
Bovine IgG1 +
Bovine IgG2 +++
Chicken IgY
Dog IgG +++
Goat IgG1 +
Goat IgG2 +++
Guinea Pig IgG +++
Hamster +
Horse IgG +
Monkey IgG +++
Porcine IgG +++
Rabbit IgG +++
Sheep IgG1 +
Sheep IgG2 +++
Table 1: Binding strength of Protein A to different species of Ig’s and their
subclasses.
4. Place the tube on the magnet. Transfer the supernatant to a clean tube for
further analysis, if desired.
5. Wash the magnetic bead-Ab-Ag complex 3 times using 200 µL Washing
Buffer for each wash. Separate on the magnet between each wash, remove
supernatant, and resuspend by gentle pipetting.
6. Resuspend the magnetic bead-Ab-Ag complex in 100 µL Washing Buffer and
transfer the bead suspension to a clean tube. This is recommended to avoid co-
elution of proteins bound to the tube wall.
Note: To store the immunoprecipitated protein, add Elution Buffer and
NuPAGE LDS Sample Buffer, then freeze the magnetic bead-Ab-Ag complex.
For subsequent analysis of the sample, thaw and proceed with step 2 of "Elute
target antigen (Denaturing elution)".
7. Proceed to “Elute target antigen”.
Elute target antigen
Denaturing elution
1. Place the tube containing the magnetic bead-Ab-Ag complex on the magnet
and remove the supernatant.
2. Add 20 µL Elution Buffer and 10 µL of pre-mixed NuPAGE LDS Sample
Buffer and NuPAGE Sample Reducing Agent.
3. Gently pipette to resuspend the magnetic bead-Ab-Ag complex.
4. Heat for 10 min at 70ºC.
5. Place the tube on the magnet and load the supernatant/sample onto a gel.
Note: As an alternative, the magnetic bead-Ab-Ag complex can be resuspended in
a sample buffer of your choice (e.g. SDS sample buffer). Follow the recommended
temperatures and heating times for these buffers prior to gel loading.
Non-denaturing elution
1. Place the tube (from step 6 "Immunoprecipitate target antigen") on the magnet
and remove the supernatant.
2. Add 20 µL Elution Buffer and gently pipet to resuspend the magnetic bead-Ab-
Ag complex. Avoid producing foam during resuspension.
3. Incubate with rotation for 2 min at room temperature to dissociate the complex.
4. Place the tube on the magnet and transfer the supernatant containing eluted Ab
and Ag to a clean tube. If the eluted protein is to be used for functional assays
or stored, the pH of the eluate can be adjusted by adding 1 M Tris, pH 7.5.
Description of materials
This product contains Dynabeads Protein A for immunoprecipitation.
DynabeadsProtein A are uniform, 2.8 μm, superparamagnetic beads with
recombinant Protein A (approximately 45 kDa) covalently coupled to the surface.
Related products
Product Cat. No.
Immunoprecipitation Kit – Dynabeads Protein G 10007D
Dynabeads Protein G 10003D
Dynabeads Protein A 10001D
DynaMag-2 magnet 12321D
HulaMixer Sample Mixer 15920D
Cell Extraction Buffer FNN0011
NuPAGE LDS Sample Buffer NP0007
NuPAGE Sample Reducing Agent NP0009
DSS (disuccinimidyl suberate) 21655
on labels is the symbol for catalog number.
REF
Limited Use Label License
These products may be covered by one or more Limited Use Label Licenses. By use
of these products, you accept the terms and conditions of all applicable Limited Use
Label Licenses.
Manufactured by Thermo Fisher Scientic Baltics UAB, V.A. Graiciuno 8, LT-02241
Vilnius, Lithuania. Thermo Fisher Scientic Baltics UAB complies with Quality
System Standards ISO 9001 and ISO 13485.
Limited product warranty
Life Technologies Corporation and/or its afliate(s) warrant their products as set
forth in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies' website at www.thermosher.com/us/en/home/global/terms-and-
conditions.html. If you have any questions, please contact Life Technologies at www.
thermosher.com/support.
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Thermo Fisher Scientific Dynabeads Protein A Immunoprecipitation Kit User guide

Type
User guide

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